The present invention relates to a development of recombinant expression vectors useful for the expression of DNA coding segments in yeasts, fungi and mammalian cells, and to the development of shuttle expression vectors, capable of expressing a DNA coding segment in both yeasts and mammalian cells. The vector system herein described employs an adenovirus promoter to control expression of the inserted DNA coding segment. The described promoter system was not previously known to be functional in non-mammalian cells.
The adenoviruses constitute a class of viruses infective to animals, including humans, and known to transform infected mammalian cells in culture with low frequency. The adenoviruses have been extensively studied and are well-characterized. The DNA nucleotide sequences are known for specific strains. (For general background, see Tooze, J., Molecular Biology of Tumor Viruses, Part II: DNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1980). Genetically, the functions of the virus are classified as "early" or "late," depending on whether they are expressed before or after the onset of viral DNA synthesis in the infection cycle. Early functions are expressed under control of several early promoters. In contrast, the majority of late functions are expressed under the control of a single, very active, promoter known as the adenovirus major late promoter (herein AML promoter). The nucleotide sequence of the AML promoter has been published, Tooze, supra. Comparison with the known nucleotide sequences of active yeast promoters reveals few points of similarity between the AML promoter and yeast promoters, other than certain short segments which appear to be characteristic of almost all eukaryotic promoters. By contrast, many points of structural similarity are known, between the yeast promoters for alcohol dehydrogenase (ADH) and glyceraldehyde phosphate dehydrogenase (GAPDH). [See Dobson, M. J., et al., Nucl. Acids Res. 10, 2625 (1982)].
A promoter is defined herein as a DNA segment capable of functioning to initiate transcription of an adjoining DNA segment. Transcription is the synthesis of RNA (herein termed messenger RNA, or mRNA), complementary to one strand of the DNA adjoining the promoter region. In eukaryotes, mRNA synthesis is catalyzed by an enzyme termed RNA polymerase II. The minimum essential elements of promoter function are two: to provide a starting point for the initiation of transcription and to provide a binding site for RNA polymerase II near the start site permitting selection of the proper strand of DNA as a template for mRNA synthesis. In addition, a eukaryotic promoter functions to regulate the relative efficiency of transcription of coding segments under its control. An active promoter is one which induces the synthesis of relatively large amounts of mRNA complementary a strand of the adjacent DNA coding segment.
The structural correlates of promoter function have not been clearly established. A promoter segment can be identified in nature as a region lying adjacent to a given structural gene at its 5' end. (References to the 5' and 3' ends of a gene will be understood to indicate the corresponding respective ends of mRNA transcribed therefrom, and these, in turn, will be understood to correlate with the NH.sub.2 -- and --COOH termini of the encoded protein, respectively. Mutations in the 5' untranslated region, adjacent to a coding segment of DNA, and extending from 200 to 400 nucleotides from the start codon thereof, display a variety of functional defects in transcription, ranging from reduced rate or efficiency of transcription, to total cessation of transcription. Where several genes are transcribed together in a single transcription unit, such mutations can result in the simultaneous loss or reduction in amount of several gene products. Such mutations define the promoter region for the structural gene or genes they affect. Comparisons of nucleotide sequences of promoters of various genes from various species have revealed only a few short regions of nucleotide sequenced similarity in common between them. Most notable of these is the "TATA box," a segment of about five to ten nucleotides located generally about 70 to 230 nucleotides upstream from the start of a coding segment, having a sequence generally resembling TATAA. For a review of structural comparisons, see Rosenberg, M. et al., Ann. Res. Genetics, 13, 319 (1979). The TATA box is believed to function in the initiation of transcription. Other examples of short regions of sequence similarity having similar locations in a number of promoters, include segments with such whimsical descriptors as "CAAT BOX," and "CACA BOX,". However, many promoters lack one or more of these features and their function is not established.
Comparative studies of the effects of promoter mutations lying distal to the start of the coding segment have been undertaken, e.g., by McKnight, S., et al., Science 217, (1982). Such studies have shown, in general, that certain regions lying upstream from the TATA box appear to be involved in the binding and orientation of the RNA polymerase to the DNA segment to be transcribed. Structural variations in this portion of the promoter presumably affect the efficiency of transcription and are known to vary substantially from one species to another.
The structures of over a dozen yeast promoters have been determined and the structures were compared by Dobson, M. J. et al., Nucleic Acids Research 10, 2625 (1982). The yeast promoters have many points of similarity not shared by non-yeast promoters, such as the adenovirus major late promoter (supra, Tooze et al.), and herpes virus thymidine kinase gene (Kiss, G., et al., J. Bact. 149, 542, 1982; Kiss, G., et al., J. Bact. 150, 465 (1982). Further, it has been shown (Kiss, G. et al., supra) that the thymidine kinase promoter of herpes virus does not function in yeast.
The present invention stems from the surprising discovery that the major late promoter of adenovirus is functional and highly active in yeast. That discovery has made it possible to construct, for the first time, expression vectors for the expression of DNA coding segments in yeast, controlled by the AML promoter, and for methods of synthesizing proteins in yeast cells transformed by such vectors.
The construction of vectors suitable for the expression of a DNA coding segment in yeast has been described, see, e.g., Ammerer, G. et al., Recombinant DNA, Proc. 3rd Cleveland Symp. Macromolecules (Walton, A. G., ed.), p. 185, Elsevier, Amsterdam (1981). Shuttle vectors, capable of replication either in a bacterial strain such as Escherichia coli and in yeast have been described. However, such vectors have relied upon the use of known yeast promoters for expression in yeast. Previously described shuttle vectors have been limited to one of the alternative hosts for expression. For example, shuttle vectors having yeast promoters are limited to expression of the DNA coding sequence in yeast only. A disadvantage of such vectors is that an extensive region of yeast homology, the promoter region, provides an opportunity for genetic recombination between the vector and the yeast chromosome, possibly resulting in integration of the vector. Consequently, the copy number per cell of sequences represented by the vector is one per chromosome. The reduction in copy number makes it impossible to achieve the highest levels of expression.
Expression vectors for yeast, containing the AML promoter, provide distinct advantages over vectors previously available in the art. In addition to promoting a high level of expression of any DNA coding sequence under AML promoter influence, such vectors, according to the present invention, lack the regions of DNA homology between vector and chromosome provided by prior art vectors employing a yeast promoter. In fact, vectors lacking any homology with yeast chromosomal DNA can be constructed, using a replication origin provided by yeast two micron circle plasmid DNA. Furthermore, the discovery of a promoter functional in both yeast and mammalian cells makes possible the construction of shuttle vectors capable of expressing a DNA coding sequence in either host. Therefore, the present invention makes possible, for the first time, the construction of true expression shuttle vectors.